Kinetic Analysis of the Effect of N‑Terminal Acetylation on Thymine DNA Glycosylase

• Published in: Biochemistry (Easton) 2022-05, Vol.61 (10), p.895-908
• Main Authors: Tarantino, Mary E., Delaney, Sarah
• Format: Article
• Language: English
• Subjects: Acetylation; DNA Repair; Fluorouracil - pharmacology; Kinetics; Lysine - metabolism; Thymine; Thymine DNA Glycosylase - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.1c00823

Thymine DNA glycosylase (TDG) is tasked with initiating DNA base excision repair by recognizing and removing T, U, the chemotherapeutic 5-fluorouracil (5-FU), and many other oxidized and halogenated pyrimidine bases. TDG contains a long, unstructured N-terminus that contains four known sites of acetylation: lysine (K) residues 59, 83, 84, and 87. Here, K to glutamine (Q) mutants are used as acetyl-lysine (AcK) analogues to probe the effect of N-terminal acetylation on the kinetics of TDG. We find that mimicking acetylation affects neither the maximal single-turnover rate k max nor the turnover rate k TO, indicating that the steps after initial binding, through chemistry and product release, are not affected. Under subsaturating conditions, however, acetylation changes the processing of U substrates. Subtle differences among AcK analogues are revealed with 5-FU in single-stranded DNA. We propose that the subtleties observed among the AcK analogues may be amplified on the genomic scale, leading to regulation of TDG activity. N-terminal acetylation, though, may also play a structural, rather than kinetic role in vivo.